DNA Recombinant Technology DNA Recombinant Technology

What and Why?What and Why?

What?: A gene of interest is inserted What?: A gene of interest is inserted into another organism, enabling it to into another organism, enabling it to be cloned, and thus studied more be cloned, and thus studied more effectivelyeffectively

Why?: Detailed studies of the Why?: Detailed studies of the structure and function of a gene at structure and function of a gene at the molecular level require large the molecular level require large quantities of the individual gene in quantities of the individual gene in pure formpure form

CloningCloning

A collection of molecules or cells, all A collection of molecules or cells, all identical to an original molecule or cellidentical to an original molecule or cell

To "clone a gene" is to make many copies of it - To "clone a gene" is to make many copies of it - for example, in a population of bacteria for example, in a population of bacteria

Gene can be an exact copy of a natural gene Gene can be an exact copy of a natural gene Gene can be an altered version of a natural gene Gene can be an altered version of a natural gene Recombinant DNA technology makes it possible Recombinant DNA technology makes it possible

Terms to KnowTerms to Know

Vector: an autonomously replicating Vector: an autonomously replicating genetic element used to carry DNA genetic element used to carry DNA fragments into a host, typically fragments into a host, typically E. coliE. coli, for , for the purpose of gene cloningthe purpose of gene cloning• Plasmid vectorPlasmid vector• Bacteriophage gamma vectorBacteriophage gamma vector

Recombinant DNA: any DNA molecule Recombinant DNA: any DNA molecule composed of sequences derived from composed of sequences derived from different sourcesdifferent sources

CleavageCleavage

It is done by Restriction enzymesIt is done by Restriction enzymes Restriction enzymes : Restriction enzymes :

Enzyme produced by bacteria that Enzyme produced by bacteria that typically recognize specific 4-8 base typically recognize specific 4-8 base pair sequences called restriction pair sequences called restriction sites, and then cleave both DNA sites, and then cleave both DNA strands at this sitestrands at this site

Inserting DNA FragmentsInserting DNA Fragments

DNA fragments are inserted into DNA fragments are inserted into vector DNA with the aid of DNA vector DNA with the aid of DNA ligasesligases

Ligases catalyze the end-to-end Ligases catalyze the end-to-end joining of DNA fragmentsjoining of DNA fragments

PlasmidsPlasmidsNaturally occurring Naturally occurring

extrachromosomal DNA extrachromosomal DNA Plasmids are circular dsDNA Plasmids are circular dsDNA Plasmids can be cleaved by restriction Plasmids can be cleaved by restriction

enzymes, leaving sticky ends enzymes, leaving sticky ends Artificial plasmids can be constructed by Artificial plasmids can be constructed by

linking new DNA fragments to the sticky linking new DNA fragments to the sticky ends of plasmid ends of plasmid

Molecular scissors; isolated from bacteria where they are used as Bacterial defense against viruses.

Molecular scalpels to cut DNA in a precise and predictable manner

Members of the class of nucleases

Restriction Enzyme

Breaking the phosphodiester bonds that link adjacent nucleotides in DNA and RNA

molecules

EndonucleaseCleave nucleic acids at internal position

ExonucleaseProgressively digest from the ends of the nucleic acid molecules

Nuclease

Endonuclease

TypeType CharacteristicsCharacteristics

II Have both restriction and modification activityHave both restriction and modification activity Cut at sites 1000 nucleotides or more away from Cut at sites 1000 nucleotides or more away from

recognition siterecognition site ATP is requiredATP is required

IIII It has only restriction site activityIt has only restriction site activity Its cut is predictable and consistent manner at a Its cut is predictable and consistent manner at a

site within or adjacent to restriction sitesite within or adjacent to restriction site It require only magnesium ion as cofactor It require only magnesium ion as cofactor

IIIIII Have both restriction and modification activityHave both restriction and modification activityCut at sites closed to recognition site Cut at sites closed to recognition site ATP is requiredATP is required

There are already more than 1200 type II enzymes isolated from prokaryotic organism

They recognize more than 130 different nucleotide sequence

They scan a DNA molecule, stopping only when it recognizes a specific sequence of nucleotides that are composed of symetrical, palindromic sequence

Palindromic sequence:The sequence read forward on one DNA strand is identical to the sequence read in the opposite direction on the complementary strand

To Avoid confusion, restriction endonucleases are named according to the following nomenclature

Restriction Enzyme

The first letter is the initial letter of the genus name of the organism from which the enzyme is isolated

The second and third letters are usually the initial letters of the organisms species name. It is written in italic

A fourth letter, if any, indicates a particular strain organism

Originally, roman numerals were meant to indicate the order in which enzymes, isolated from the same organisms and strain, are eluted from a chromatography column. More often, the roman numerals indicate the order of discovery

Nomenclature

NomenclatureEcoEcoRIRI E : Genus EscherichiaE : Genus Escherichia

co: Species colico: Species coli

R : Strain RY13R : Strain RY13

I : First endonuclease isolatedI : First endonuclease isolated

BamBamHIHI B : Genus BacillusB : Genus Bacillus

am: species amyloliquefaciensam: species amyloliquefaciens

H : Strain HH : Strain H

I : First endonuclease isolatedI : First endonuclease isolated

HinHindIIIdIII H : Genus HaemophilusH : Genus Haemophilus

in : species influenzaein : species influenzae

d : strain Rdd : strain Rd

III : Third endonuclease isolatedIII : Third endonuclease isolated

SpecificityEnzymeEnzyme SourceSource SequenceSequence EndEnd

BamHIBamHI Bacillus amyloliquefaciens Bacillus amyloliquefaciens HH

GGGATCCGATCC StickStickyy

BglIIBglII Bacillus globigiiBacillus globigii AAGATCTGATCT StickStickyy

EcoRIEcoRI Escherichia coli RY13Escherichia coli RY13 GGAATTCAATTC StickStickyy

EcoRIIEcoRII Escherichia coli R245Escherichia coli R245 CCTGGCCTGG StickStickyy

HaeIIIHaeIII Haemophilus aegyptiusHaemophilus aegyptius GGGGCCCC BluntBlunt

HindIIHindII Haemophilus influenzae Haemophilus influenzae RdRd

GTPyGTPyPuACPuAC BluntBlunt

HindIIIHindIII Haemophilus influenzae Haemophilus influenzae RdRd

AAAGCTTAGCTT StickStickyy

HpaIIHpaII Haemophilus Haemophilus parainfluenzaeparainfluenzae

CCCGGCGG StickStickyy

NotINotI Nocardia otitidis-caviarumNocardia otitidis-caviarum GCGCGGCCGCGGCCGC StickStickyy

PstIPstI Providencia stuartii 164Providencia stuartii 164 CTGCACTGCAGG StickStickyy

Restriction enzymesRestriction enzymes

degenerate or specific sequences

kind of ends produced (5’ or 3’ overhang (sticky), blunt)

number of nucleotides recognized (4, 6,8 base-cutters most common)

Become familiar with the back of your molecular biology catalog!

whether cleavage occurs within the recognition sequence

Restriction enzymes can be grouped by:

A restriction enzyme (A restriction enzyme (EcoEcoRI)RI)

1. 6-base cutter

4. produces a 5’ overhang (sticky end)

2. Specific palindromic sequence (5’GAATTC)

3. Cuts within the recognition sequence (type II enzyme)

Restriction enzymesRestriction enzymes

Cloning VectorsCloning Vectors

Plasmids that can be modified to carry Plasmids that can be modified to carry new genes new genes

Plasmids useful as cloning vectors must have Plasmids useful as cloning vectors must have • aa replicator replicator (origin of replication) (origin of replication) • a a selectable markerselectable marker (antibiotic resistance (antibiotic resistance

gene) gene) • a a cloning sitecloning site (site where insertion of (site where insertion of

foreign DNA will not disrupt replication or foreign DNA will not disrupt replication or inactivate essential markersinactivate essential markers

A typical plasmid vector with a A typical plasmid vector with a polylinkerpolylinker

Chimeric PlasmidsChimeric Plasmids

Named for mythological beasts with Named for mythological beasts with body parts from several creaturesbody parts from several creatures

After cleavage of a plasmid with a After cleavage of a plasmid with a restriction enzyme, a foreign DNA restriction enzyme, a foreign DNA fragment can be inserted fragment can be inserted

Ends of the plasmid/fragment are closed Ends of the plasmid/fragment are closed to form a "to form a "recombinant plasmidrecombinant plasmid" "

Plasmid can replicate when placed in a Plasmid can replicate when placed in a suitable bacterial host suitable bacterial host

Directional CloningDirectional Cloning

Often one desires to insert foreign DNA Often one desires to insert foreign DNA in a particular orientationin a particular orientation

This can be done by making two This can be done by making two cleavages with two different restriction cleavages with two different restriction enzymes enzymes

Construct foreign DNA with same two Construct foreign DNA with same two restriction enzymes restriction enzymes

Foreign DNA can only be inserted in one Foreign DNA can only be inserted in one direction direction